Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae

Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.• Buffers were redesigned from [32,33,35].• No DNASE, Ribonuclease A or beads were used during the purification.• Simple and inexpensive dsRNA extraction and purification method is described.

DsRNA isolation is, therefore, an essential step to study these processes and to identify dsRNA viruses.The ability to isolate dsRNA is a valuable tool to investigate the viral diversity and evolution [14] .
The most used methods of dsRNA isolation use fibrous or microgranular cellulose.These methods rely on the affinity of cellulose powder specifically for nucleic acids, followed by dsRNA adsorption with an ethanol concentration of 15 %, and then, release of the dsRNA from cellulose without ethanol [15][16][17][18][19][20][21] .Other methods use Hydroxyapatite alone [22][23][24] or in combination with cellulose [25] , differential precipitation of ssRNA and dsRNA with Lithium chloride [26] or various other types of resins [27][28][29][30] .In these methods, an initial nucleic acid extraction is done, mainly with phenol (pH 7), followed by chromatographic separation to obtain dsRNA.All these methods are time-consuming or expensive.
Furthermore, for the initial cell lysis step these methods generally used enzymes or beads [31] , which can be expensive or require specialized equipment, respectively.An alternative method for cell pre-treatment, consists of washing with EDTA, followed by an incubation in Tris-H 2 SO 4 buffer with 2-mercaptoethanol ( ME) before phenol extraction [32] .
Nucleic acids can be partitioned between the aqueous or the phenol phase according to the pH.For example, phenol equilibrated with a pH 4 buffer can be used to remove DNA from the aqueous phase [33 , 34] .Furthermore, according to Flegr [33] , extraction buffer with ammonium sulphate (AMS) saturation between 20 to 60 % allows ssRNA removal to the phenolic phase.In addition to the phenol-based methods, a total RNA precipitation method has been described, in which DNA remains in solution in the supernatant by adding 0.5 vol ammonium acetate 7.5 M to the total nucleic acid sample [35] .For a cheaper, simpler, and faster dsRNA isolation method we propose an approach based on the solubility of both DNA and ssRNA compared to dsRNA in the phenol phase.
Hereby is presented an optimized method for isolating dsRNA from yeast, as demonstrated in a Saccharomyces cerevisiae strain.The approach is a sequential combination and adaptation of the following techniques from previous studies: pre-treatment of S. cerevisiae cells with ME [32] , the use of pH 4 phenol [33 , 34] and addition of AMS to remove ssRNA [33] in the extraction method, and the use of ammonium acetate to precipitate total RNA whilst leaving the DNA in the supernatant [35] .
The protocol relies on partitioning DNA and ssRNA into the phenolic phase, facilitated by low pH and the presence of AMS in the extraction buffer, respectively, while dsRNA is left in the supernatant.To precipitate the dsRNA from the supernatant, an appropriate volume of ammonium acetate is added to the sample.This new isolation method was thoroughly validated by comparison with the standard fibrous cellulose dsRNA isolation method, followed by an RT-PCR using novel primers targeting the dsRNA L-BC virus from S. cerevisiae , and confirmation through Sanger DNA sequencing.
This optimized dsRNA isolation method, which is cheaper, faster, and simpler, has great potential for advancing research in molecular virology and to be applied in dsRNA-based studies in general.4) or with TE (pH 7) a total of 3 times.5. Add 0.1 % 2-Mercaptoethanol to the aqueous phase and mix.Allow the phases to separate.6.The buffer saturated phenol may be stored at 4 °C for periods up to 5 months.

Cell wall treatment
To avoid the use of enzymes or beads, we used a ME-based method [32] but increased the ME concentration to 5 % and the incubation time to 40 min to ensure efficient cell wall break.After incubation, the cells were collected by centrifugation at 6,000 g/10 min, the supernatant was discharged, and the cell pellet was weighed.The cells were then resuspended in 50 mM Na 2 EDTA (pH 8.0), and pelleted again to remove EDTA.Cell pellets were resuspended and incubated for 40 min in 50 mM Tris-H 2 SO 4 (pH 9.3) with 5 % ME, and then centrifuged at 10,000 g for 10 min.Cell pellets, of approximately 50 mg, were used immediately or maintained at -80 °C until further use.

Total nucleic acid extraction
This method [32] , with modifications, was used to confirm the total nucleic acids present in the sample and can be used as a first step for the dsRNA isolation methods.

Table 1
Comparison between the various described dsRNA purification methods.

Phases of the method Methods
Total nucleic acids [32] Cellulose [18] Flag method [33  12. Centrifuge at 14,000 g for 15 min at 4°C to pellet the precipitated dsRNA.
NOTE: this must be performed at 4°C, to ensure good RNA precipitation.
14. Centrifuge at 10,000 g for 5 min and discard the supernatant by decanting and add 500 μl 100 % ethanol, to wash the dsRNA pellet from any traces of phenol.15.Dry at RT and resuspend in 50 μl STE 1 X buffer and maintain the sample at -20°C.
Table 1 summarizes the comparison between the various methods used for dsRNA purification and the alternative method presented in this article.

Qualitative and quantitative analyses of the isolated dsRNA
A simple, rapid, and relatively inexpensive spectrophotometric assay was performed to assess the purity of the extracted dsRNA.Ratios of UV absorption at A260/280 and A260/230 were recorded using Nano-Drop ND-1000 (Thermo-Scientific, USA).Nucleic acid extraction was assessed by agarose gel electrophoresis.DsRNA concentration was determined according to the extinction coefficient of 46 μg/mL/A260 [36 , 37] .

Validation of RNA quality and integrity using PCR amplification and DNA sequencing
To validate the extracted RNA quality, a fragment from the Viral RNA-directed RNA-polymerase from L-BC Totivirus was amplified using a One-tube RT-PCR [38] with modifications described below.

Gel electrophoresis
Ten (10) μl of dsRNA extraction or PCR product were subjected to electrophoresis on a 0.5 mg/ml ethidium bromide (Sigma) stained 1 % (w/v) agarose (Fisher Scientific, USA) gel in TAE buffer.The gels were analyzed and documented on DigiGenius Gel Doc (Syngene, USA).

DNA sequencing
After electrophoresis at 100 V for 45 min on a 1 % agarose gel in TAE buffer, the band of the desired size was cut from the gel and the DNA was isolated with the MicroElute -Gel Extraction kit (#D6294-01, Omega, USA), according to the manufacturer's instructions.The collected DNA was sequenced by the Sanger method (StabVida, Portugal), using the PCR primers.

Method validation
The yeast cells were treated to break the cell walls using the technique described by Fried and Fink [32] , modified by increasing the amount of ME from 2.5 % to 5 % and extending the incubation time from 15 min to 40 min for greater efficiency.Nucleic acids were purified using the "Total nucleic acids extraction from yeast" protocol and include the dsRNA plasmid 4.6 kbp expected band [4 , 6] .The extration was made in duplicate from the same cultured sample 1 and 2 in ( Fig. 1 ).
To remove both DNA and ssRNA from the nucleic acid solution, we made modifications to the method described by Flegr [33] .The original protocol included the addition of solid AMS to the sample before extraction, but this approach is time-consuming and can be susceptible to contamination.To enhance this protocol, we evaluated incorporation of AMS directly into the extraction buffer.We tested various concentrations of AMS in the extraction buffer (from 5 to 25 % w/v), two phenol pH levels (pH 4 and pH 7), and two incubation temperatures (25°C and 60°C).The optimal AMS concentration was found to be 20 % (w/v), as at greater concentrations dsRNA entered the phenol phase (Supplementary information Fig. 1).However, at 20 % AMS (w/v) the phenol and aqueous phases are inverted, as previously reported by Flegr [33] .To overcome this phenomenon, we added CIA into phenol to a ratio of 10:1.This phenol/CIA mixture, ensured that the aqueous phase remained at the top.We confirmed that 20 % w/v AMS in the buffer separates ssRNA into the phenol phase while preserving dsRNA in the aqueous phase as previously reported by Flegr [ 33 ] by gel electrophoresis, as ssRNA become undetectable in the aqueous phase.However, it should be noted that more than two phenol extractions result in dsRNA also being removed into the phenol phase.To effectively remove any DNA that might be present in the sample, the phenol phase should have a low pH.Here, phenol equilibrated with Sodium Acetate at pH 4 [33 , 34] and phenol at an initial pH 7 produced similar results ( Fig. 2 ), which could be due to the extraction buffer having a final pH of 4, after the addition of AMS, thus reducing the phenol pH.The protocol for dsRNA extraction with best results was: SS20 buffer, 1 st phenol:CIA extraction at 60°C, for 30 min and 2 nd phenol:CIA at RT.Both ssRNA and DNA were removed from the sample to undetectable levels in gel electrophoresis, but the presence of DNA or ssRNA was not tested by other means.
To precipitate dsRNA, the method described by Flegr [33] was challenging to perform.Instead, we tested adding ammonium acetate solution, which is commonly used for total RNA precipitation [35] .Ammonium acetate volumes from 0.5 vol [35] to 1 vol  (3.75 M final concentration) were equally effective at DNA removal, as it remained in the supernatant according to Knapp [ 35 ].However, the higher volume (1 vol) resulted in a higher yield of dsRNA recovered after precipitation (Supplementary information -Fig.2).We observed no interference from the AMS already present in the buffer, regarding DNA removal or dsRNA yield.This protocol using 1 vol (3.75 M final concentration) ammonium acetate is an efficient and easy to perform alternative method for total RNA precipitation even in the presence of AMS.
The obtained dsRNA yield and purity, using our adapted combined cell wall treatment-extraction-precipitation method, is comparable to the traditional method used for dsRNA purification, which relies on cellulose ( Fig. 2 and Table 3 ) However, a lower 260/230 ratio was observed after using the SS20 buffer, which could be due to low SDS concentration (0.2 % w/v) used in the extraction buffer.
To ensure the presence and quality of the extracted dsRNA, we used one tube RT-PCR amplification of the viral gene RNAdirected RNA-polymerase from L-BC Totivirus, using primers developed by us.For successful amplification, we found that a prior dsRNA denaturation step with DMSO at 95 °C for 1 min was necessary before adding it to the RT-PCR mix.Without this step, or with different temperatures and incubation times (37°C for 30 min or 65°C for 20 min), no RT-PCR amplification was observed.The dsRNA samples isolated using the different methods, including the new phenol partition method with an AMS buffer, were equally Note : Results are expressed as the mean of 3 samples ± standard deviation.amenable to successful RT-PCR amplification with L21/L22 primers, producing the expected 650 bp amplicon with similar band intensity ( Fig. 3 ).The amplicon was sequenced using Sanger sequencing, yielding a high-quality sequence (Supplementary information-Fig.3).Upon a BLASTn search against the National Center for Biotechnology Information (NCBI) database, this sequence was found to have 97.64 % similarity to the previously reported S. cerevisiae L-BC virus sequence (accession NC_001641.1),and a BLASTp search gave 95.44 % similarity with S. cerevisiae PYCC 3938 strain that harbors Totivirus L-BC-2 virus (accession APR62628.1).The cDNA sequence has been deposited in GenBank under accession number OR393309.
We describe, here, a new method for dsRNA extraction and purification that, in summary, combines an initial cell wall treatment with 5 % ME, AMS at 20 % in the extraction buffer for the phenol:CIA (10:1) extraction step and ammonium acetate (1vol 7.5 M) for dsRNA precipitation.
The methods described by Fleg [33] for dsRNA isolation primarily focus on low pH buffer and on the use of ammonium sulfate added to the extraction buffer after an initial extraction, and they are specifically applied to Trichomonas sp.dsRNA isolation.Our method, dsRNA isolation -phase partition, offers a more practical and applicable approach to isolating dsRNA in S. cerevisiae .It allows the removal of both DNA and ssRNA into the phenol phase using a single buffer, facilitated by maintaining a low pH (4) and incorporating AMS in the aqueous phase.
Another improvement in our method was the addition of chloroform to the extraction buffer, which facilitated dsRNA extraction and prevented the inversion of the aqueous/phenol phases due to the increased density resulting from the addition of ammonium sulfate in the buffer.
Moreover, our method yields similar or even better results in terms of the quantity of isolated dsRNA when compared to the traditional cellulose-based dsRNA methods [15][16][17][18][19][20][21] .Despite lower 260/230 nm rations, the dsRNA obtained was of sufficiently good quality and quantity for RT-PCR amplification.In relation to the cellulose method of dsRNA purification, our method offers several advantages.It reduces the number of extraction and purification steps and cuts down the purification time from 4 h to 2 h.
We not only were able to isolate high-quality genomic dsRNA in yeast, but we also demonstrated that it is well-suited for amplification followed by sequencing.Further tests of this method will include direct RNA sequencing applications.

Conclusion
The improved method for genomic viral dsRNA isolation presented here addresses a crucial aspect of virology research, by offering a practical and efficient approach to sample preparation, suitable for various molecular analytical techniques and dsRNA amplification, including the detection and discovery of dsRNA virus in pathogens and their hosts (work in progress).
Our approach stands out from the numerous methods published for dsRNA extraction, for its low cost, simplicity, reliability, and reproducibility, making the extraction of high-quality dsRNA from yeast samples more available for low-technology laboratories.

Table 2
Primers used in the study.
10. Carefully transfer the aqueous upper phase to a new tube and add 1 vol ammonium acetate (7.5M).11.Gently invert 5-6 times and then incubate at -20 °C for 30 min to precipitate the dsRNA.

Table 3
DsRNA concentrations obtained with the various methods.